Gene replacement therapy has great potential to alleviate the prognosis of many incurable neurodegenerative diseases. In this approach, a defective gene is replaced in situ by a gene of which its products can restore normal function. Up to date there is no direct method to monitor non-invasively the accuracy of the gene delivery and it's expression level for prolonged time periods and co-register it with anatomical structures in the brain. The main goal of the current proposal is to develop a methodology for the non-invasive imaging of gene delivery to the central nervous system (CNS) based on artificial Magnetic Resonance Imaging (MRI) reporter genes such as the Lysine Rich Protein (LRP) reporter gene, which we have previously developed for tracking cells. This approach is based on the chemical exchange saturation transfer (CEST) contrast mechanism. A radiofrequency pulse is applied to saturate specific amide protons, reducing the water signal. Since different exchangeable protons have different resonance frequencies, this allows creation of a family of reporter genes that are distinguishable from each other in a frequency-selective manner. To test our hypothesis that transgenic expression can be monitored directly in the rat brain with a CEST reporter gene, viral delivery and expression of LRP will be monitored using CEST MRI and will be validated by bioluminescence imaging using luciferase. Next, two libraries of reporter genes having different radiofrequency selectivity will be generated. One library will contain artificial genes of the LRP-type, and the other will contain genes that are similar to protamine (a protein with high arginine concentration). The libraries will be screened for optimized reporter genes. Since the brain is a heterogeneous tissue, to further test our hypothesis it is imperative to image gene expression in a broad range of different cell types. To this end, lentiviruses expressing the Vesicular System Stomatitis Virus G glycoprotein (VSV-G) will be used as a shuttle vector, with gene expression under cell-specific promoters (NSE for neurons and GFAP for astrocytes). Cell-specific gene expression will be assessed by CEST MRI in vivo in rats in a frequency- selective manner and will be validated with histology. Due to the capacity of the lentivirus to carry therapeutic genes in addition to the reporter gene, we anticipate that our approach is applicable for real-time monitoring of the efficiency, safety, and levels of gene expression in gene replacement therapy. Many neuropathological processes are complex and frequently require the replacement of more than one gene, occasionally even in multiple cell types. Thus, imaging multiple genes simultaneously in a non-invasive, serial manner may greatly aid monitoring the outcome of gene replacement therapy. PUBLIC HEALTH RELEVANCE: Gene replacement therapy has great potential to alleviate the prognosis of many incurable neurodegenerative diseases. The main goal of the current proposal is to develop a methodology for the non-invasive imaging of gene delivery to the central nervous system (CNS) using artificial reporter genes designed specifically for Magnetic Resonance Imaging (MRI). This new technology should be applicable for real-time monitoring of the efficiency, safety and levels of gene expression in gene replacement therapy. The benefits of this novel imaging approach could be further expanded to different organs and variety of applications, such as monitoring cell survival in response to cell transplantation or drug treatment.